Designing the antimicrobial peptide with centrosymmetric and amphipathic characterizations for improving antimicrobial activity.

Antibiotic-resistant bacterial infections are becoming a serious health issue and will cause 10 million deaths per year by 2050. As a result, the development of new antimicrobial agents is urgently needed. Antimicrobial peptides (AMPs) are found in the innate immune systems of various organisms to effectively fend off invading pathogens. In this study, we designed a series of AMPs (THL-2-1 to THL-2-9) with centrosymmetric and amphipathic properties, through substituting different amino acids on the hydrophobic side and at the centrosymmetric position to improve their antimicrobial activity. The results showed that leucine as a residue on the hydrophobic side of the peptide could enhance its antimicrobial activity and that glutamic acid as a centrosymmetric residue could increase the salt resistance of the peptide. Thus, the THL-2-3 peptide (KRLLRELKRLL-NH2 ) showed the greatest antimicrobial activity (MIC90 of 16 µM) against Gram-negative bacteria and had the highest salt resistance and cell selectivity among all the designed peptides. In summary, the results of this study provide useful references for the design of AMPs to enhance antimicrobial activity.

Structure activity relationships of peptidic analogs of MyoD for the development of Id1 inhibitors as antiproliferative agents.

Id proteins, inhibitors of DNA binding proteins, have highly conserved dimerization motif known as the helix-loop-helix (HLH) domain that acts as a negative regulator of basic HLH (bHLH) transcription factors. In signaling pathways, Id proteins play an important role in cellular development, proliferation, and differentiation. The mechanism of Id proteins is to antagonize bHLH proteins, thereby preventing them from binding to DNA and inhibiting transcription of cellular differentiation-associated genes in cancer. Recently, we reported an inhibitor of Id1, peptide 3C, which showed good affinity to Id1 protein and exhibited inhibitory effects in cancer cells. In this study, Ala (A)-substituted analogs of peptide 3C were synthesized by SPPS, purified by RP-HPLC, and characterized by MALDI-TOF MS. Binding of each peptide to Id1 or Id1-HLH (the HLH domain of Id1) was monitored by surface plasmon resonance (SPR)-based biosensor. Biological effect of each peptide in MCF-7 breast cancer cells was analyzed by MTT cell viability assay. The secondary structure of substituted analogs of peptide 3C was investigated by circular dichroism (CD) spectroscopy. SPR results revealed that A-substituted analogs of peptide 3C showed weaker binding to Id1 than that of peptide 3C, indicating that the six amino acid residues in the N-terminal of peptide 3C were all essential for binding to Id1 and the importance of amino acid residue was I(2) > Q(6) > Y(1) > G(4) > L(5) > E(3). In addition, substitution of E(3) in peptide 3C with D, Q, and R did not improve the binding potency of peptide 3C. MTT assay demonstrated that neither A-substituted nor position 3-substituted analogs of peptide 3C showed increased antiproliferative effect in MCF-7 cancer cells. CD results indicated that peptide 3C exhibited the highest content of α-helical structure (39.37%), suggesting that the α-helical structure may contribute to its binding potency for Id1 and Id1-HLH. SAR results provided important information for the development of peptidic inhibitors of Id1 as anticancer agents and demonstrated peptide 3C as a promising lead for further modifications.

Anticancer activities of an antimicrobial peptide derivative of Ixosin-B amide.

In nature, antimicrobial peptides (AMPs) represent the first line of defense against infection by pathogens; thus, they are generally good candidates for the development of antimicrobial agents. Recently, we reported two potent antimicrobial peptides, KWLRRVWRWWR-amide (MAP-04-03) and KRLRRVWRRWR-amide (MAP-04-04), which were derived from a fragment of Ixosin-B-amide (KSDVRRWRSRY). Since some cationic AMPs exhibited cytotoxic activity against cancer cells, in the current study, we further investigated the anticancer activity of these potent antimicrobial peptides by antiproliferative assays and wound-healing assays, and the effect of peptide on the cytoskeleton alteration and cell morphology were analyzed by confocal microscopy. Results indicated that MAP-04-03 not only exhibited inhibitory effects on the proliferation (IC50=61.5 µM) and on the cell migration of MCF-7 breast cancer cells (at a concentration of 5 µM), but also affected the cytoskeleton at the concentration of 25 µM. These results demonstrated that MAP-04-03 can serve as a lead peptide analog for developing potent anticancer agents.

Structure-activity relationship of potent antimicrobial peptide analogs of Ixosin-B amide.

There is a great urgency in developing a new generation of antibiotics and antimicrobial agents since the bacterial resistance to antibiotics have increased dramatically. A series of overlapped peptide fragments of Ixosin-B, an antimicrobial peptide with amino acid sequence of QLKVDLWGTRSGIQPEQHSSGKSDVRRWRSRY, was designed, synthesized and examined for their antimicrobial activities against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. A potent 11-mer peptide TSG-8-1, WWSYVRRWRSR-amide, was developed, which exhibited antimicrobial activity against E. coli and S. aureus while very little hemolytic activity in human erythrocytes was observed at high dose level. This peptide could be further modified for the development of a potent antimicrobial agent in the future.

Discovery of potent antimicrobial peptide analogs of Ixosin-B.

Antimicrobial peptides (AMPs) represent the first defense line against infection when organisms are infected by pathogens. These peptides are generally good targets for the development of antimicrobial agents. Peptide amide analogs of Ixosin-B, an antimicrobial peptide with amino acid sequence of QLKVDLWGTRSGIQPEQHSSGKSDVRRWRSRY, were designed, synthesized and examined for antimicrobial activities against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Within the peptides synthesized, we discovered an 11-mer peptide, KRLRRVWRRWR-amide, which exhibited potent antimicrobial activity while very little hemolytic activity in human erythrocytes was observed even at high dose level (100 µM). With further modifications, this peptide could be developed into a potent antimicrobial agent in the future.

Structure-activity relationships of a peptidic antagonist of Id1 studied by biosensor method, circular dichroism spectroscopy, and bioassay.

Id1 proteins, inhibitors of differentiation or DNA binding, act as dominant negative antagonists of the bHLH family of transcription factors, which play an important role in cellular development, proliferation, and differentiation. The mechanism of Id proteins is to antagonize bHLH proteins by forming high-affinity heterodimers with other bHLH proteins, thereby preventing them from binding to DNA and inhibiting transcription of differentiation-associated genes. Our goal is to study the SARs of a peptidic antagonist of Id1, peptide 3C, which exhibits high affinity for Id1 and inhibitory effect on the proliferation of cancer cells. A series of N-terminal- and C-terminal-deleted analogs of peptide 3C were designed, synthesized, and characterized. Affinity of each peptide for Id1 or Id1-HLH domain was determined by SPR-based biosensor. The secondary structure of each peptide was studied by CD spectroscopy. Biological effect of each peptide in breast cancer cell (MCF-7) was analyzed by the MTT cell viability assay. Results demonstrated that peptide 3C and peptide 3C-CtD4 exhibited higher affinity for Id1-HLH and the equilibrium dissociation constants (K(D) ) were 3.16 and 2.77 µM, respectively. CD results indicated that the percentage of α-helix (%) in the secondary structure of deleted peptides were different, ranging from 7.93 to 10.45%. Although MTT results showed that treatment of MCF-7 with these peptides did not cause antiproliferative effects in cancer cells, SPR results demonstrated that the high-affinity peptides 3C and 3C-CtD4 are promising for further modifications to enhance their affinity for Id1-HLH and antiproliferative effects in cancer cells and for the development of peptidic antagonists as anticancer agents.

Affinity of synthetic peptide fragments of MyoD for Id1 protein and their biological effects in several cancer cells.

MyoD is a DNA-binding protein capable of specific interactions that involve the helix-loop-helix (HLH) domain. The HLH motif of MyoD can form oligomers with the HLH motif of Id1 (the inhibitor of DNA-binding proteins) that folds into a highly stable helical conformation stabilized by the self-association. The Id family consists of four related proteins that contain a highly conserved dimerization motif known as the HLH domain. In signaling pathways, Id proteins act as dominant negative antagonists of the basic helix-loop-helix (bHLH) family of transcription factors which play important roles in cellular development, proliferation, and differentiation. The mechanism of Id proteins is to antagonize bHLH proteins by binding as dominant negative HLH proteins to form high-affinity heterodimers with other bHLH proteins, thereby preventing them from binding to DNA and inhibiting transcription of differentiation-associated genes. The goal of this study is to design and synthesize peptide fragments of MyoD with high affinity for Id1 to interrupt the interactions among Id1, MyoD, and other bHLH DNA-binding proteins and to inhibit the proliferation of cancer cells. Affinity of each peptide for Id1 was determined by surface plasmon resonance (SPR) technology. The secondary structure of each peptide was studied by circular dichroism (CD) spectroscopy. Biological effects of each peptide in several cancer cells such as breast and colon cancer cells were analyzed. Results demonstrated that the peptide 3C (H-Tyr-Ile-Glu-Gly-Leu-Gln-Ala-Leu-Leu-Arg-Asp-Gln-NH(2)) not only showed high affinity for Id1 but also exhibited antiproliferative effects in HT-29 and MCF-7 cancer cells; the IC(50) value of 3C was determined as 25 microM in both cells. The percentage of sub-G1 in the cell cycle of the cancer cells treated with 5 microM of 3C was increased, indicating the induced apoptosis of cancer cells by 3C. Taken together, the peptide 3C is a promising lead compound for the development of antiproliferative agents.

Effects of peptidic antagonists of Grb2-SH2 on human breast cancer cells.

The growth factor receptor-bound protein Src homology 2 (Grb2-SH2) plays an important role in the oncogenic Ras signaling pathway, which involves in cell proliferation and differentiation. Therefore, the antagonist of Grb2-SH2 has become a potential target for developing anticancer agents. Recently, we discovered a peptide 1 (Fmoc-Glu-Tyr-Aib-Asn-NH(2)) with high affinity for the Grb2-SH2 domain by using surface plasmon resonance (SPR)-biosensor technology. Herein, we report the further design of the lead peptide 1 by addition of an Arg-Gly-Asp sequence to 1 to enhance binding to Grb2-SH2 and inducing apoptosis in cancer cells. Both the linear and cyclic analogs of the newly designed compound were prepared along with an analog in which the N(alpha)-Fmoc group was removed. These peptide analogs were assayed for their affinity for the Grb2-SH2, their antiproliferative effect on human breast cancer cells, their specificity for cancer cells, and their effects on cytotoxicity and the cell cycle. MCF-7 and MDA-MB-453 breast cancer cells were treated with various concentrations of each peptide. The cell viability and cytotoxicity of peptide-treated cells were determined by using the cell proliferation kit (3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-tetrazolium bromide, MTT) and cytotoxicity kit (lactate dehydrogenase, LDH), respectively. Effects of peptides on the cell cycle progression of cancer cells and apoptosis were analyzed by using flow cytometry. Results demonstrated that the peptide analog 2 (H-Arg-Gly-Asp-Glu-Tyr-Aib-Asn-Arg-Gly-Asp-NH(2)) had anti-proliferative effects on MCF-7 and MDA-MB-453 cells with an IC(50) of 45.7 microM and 47.4 microM, respectively. The cytotoxicity and percentage of sub-G1 in the cell cycle were increased in these cancer cells when cells were treated with higher concentration of the Arg-Gly-Asp-containing peptide 2. These results provide important information for the development of anti-cancer agents.

Determination of binding potency of peptidic inhibitors of Grb2-SH2 by using the protein-captured biosensor method.

The growth factor receptor-binding protein 2-Src homology 2 (Grb2-SH2) domain plays an important role in the oncogenic Ras signal transduction pathway, therefore, peptidic inhibitors of the Grb2-SH2 domain has been chosen as our target for the development of antiproliferative agents. The inhibitory effects of peptide analogs on the Grb2-SH2 domain have been determined by using surface plasmon resonance (SPR) technology developed with the BIACORE biosensor. Recently, we reported the analysis of interactions between peptides and the GST-Grb2-SH2 that was immobilized on the surface of sensor chip by using BIACORE biosensor (the protein-immobilized method). Herein, we analyze interactions of peptides with the GST-Grb2-SH2 that was captured by the anti-GST antibodies immobilized on the surface of sensor chip (the protein-captured method). Results obtained by both methods are in good correlation, indicating the immobilization of GST-Grb2-SH2 on the sensor chip did not significantly affect the binding of Grb2-SH2 with peptides. Both SPR-based assays are very sensitive bioanalytical methods and can be applied in screening inhibitors of target proteins or purifying GST-fusion proteins, however, considering the efficiency and the cost, the GST-Grb2-SH2-immobilized method is suggested for routinely determining the binding potency of inhibitors of Grb2-SH2.

Small nonphosphorylated Grb2-SH2 domain antagonists evaluated by surface plasmon resonance technology.

The growth factor receptor-binding protein-Src homology 2 (Grb2-SH2) domain plays an important role in the oncogenic Ras signal transduction pathway, which involves cell proliferation and differentiation. Therefore, the Grb2-SH2 domain has been chosen as our target for development of potential antiproliferative agents. Herein, we report the study of the inhibitory effects of small nonphosphorylated peptide analogs interacting with the Grb2-SH2 domain protein by surface plasmon resonance (SPR) technology. A set of 8 related peptide analogs were synthesized, purified, and characterized. Their inhibitory effects on Grb2-SH2 were evaluated by the SPR technology developed with the BIACORE X instrument. The lead peptide, Fmoc-Glu-Tyr-Aib-Asn-NH2 (Fmoc-E-Y-Aib-N; Fmoc: 9-fluorenylmethyoxycarbonyl; Aib=alpha-amino isobutyric acid) inhibited Grb2-SH2 domain function with an IC50 value of 8.7 microM. A molecular modeling study of the lead peptide indicated that the glutamate in the Fmoc peptide is ideally positioned to form a strong salt bridge to Arg 67 in the Grb2-SH2 domain, using both its backbone carbonyl and its acidic group. Residue Glu 89 in Grb2-SH2 flips inward to fill the binding site and partially replace the phosphate group as a hydrogen-bond acceptor. Results of these studies provide important information for further development of potent nonphosphorylated peptide inhibitors of the Grb2-SH2 domain.

Global optimization of conformational constraint on non-phosphorylated cyclic peptide antagonists of the Grb2-SH2 domain.

Following our earlier work on a phage library derived non-phosphorylated thioether-cyclized peptide inhibitor of Grb2 SH2 domain, a series of small peptide analogues with various cyclization linkage or various ring size were designed and synthesized and evaluated to investigate the optimal conformational constraint for this novel Grb2-SH2 blocker. Our previous SAR studies have indicated that constrained conformation as well as all amino acids except Leu(2) and Gly(7) in this lead peptide, cyclo(CH(2)CO-Glu(1)-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr-Cys(10))-amide (termed G1TE), was necessary for sustenance of the biological activity. In this study, in an effort to derive potent and bioavailable Grb2-SH2 inhibitor with minimal sequence, we undertook a systematic conformational study on this non-phosphorylated cyclic ligand by optimizing the ring linkage, ring configuration and ring size. The polarity and configuration of the cyclization linkage were implicated important in assuming the active conformation. Changing the flexible thioether linkage in G1TE into the relatively rigid sulfoxide linkage secured a 4-fold increase in potency (4, IC(50)=6.5 microM). However, open chain, shortening or expanding the ring size led to a marked loss of inhibitory activity. Significantly, the introduction of omega-amino carboxylic acid linker in place of three C-terminal amino acids in G1TE can remarkably recover the apparently favorable conformation, which is otherwise lost because of the reduced ring size. This modification, combined with favorable substitutions of Gla for Glu(1) and Adi for Glu(4) in the resulting six-residue cyclic peptide, afforded peptide 19, with an almost equal potency (19, IC(50)=23.3 microM) relative to G1TE. Moreover, the lipophilic chain in omega-amino carboxylic acid may confer better cell membrane permeability to 19. These newly developed G1TE analogues with smaller ring size and less peptide character but equal potency can serve as templates to derive potent and specific non-phosphorylated Grb2-SH2 antagonists.

Monitoring bone loss using ELISA and surface plasmon resonance (SPR) technology.

Osteoporosis influences the health of the females who are in menopause phase. The techniques to detect the markers of bone turnover is very important for preventing osteoporosis. ELISA was developed for detection of urinary N-telopeptide (NTx) as an osteoporosis marker. Our aim is to develop a sensitive method to detect NTx excretion using surface plasmon resonance (SPR). Samples collected were assayed and results suggest that our SPR-based method is promising for monitoring bone loss.

Grb2 SH2 domain-binding peptide analogs as potential anticancer agents.

The growth factor receptor-bound protein 2 (Grb2) plays an important role in the Ras signaling pathway. Several proteins were found to be overexpressed by oncogenes in the Ras signaling pathway, rendering Grb2 a potential target for the design of antitumor agents. Blocking the interaction between the phosphotyrosine-containing activated receptor and the Src-homology 2 (SH2) domain of Grb2 thus constitutes an important strategy for the development of potential anticancer agents. X-ray, NMR structural investigations, and molecular modeling studies have provided the target structure of Grb2 SH2 domain-alone or complexed with a phosphotyrosine-containing peptide-which is useful for the structure-based design of peptides or peptidomimetics with high affinity for the Grb2 SH2 domain. We review here the variety of approaches to Grb2 SH2 pepide inhibitors developed with the aim of interrupting Grb2 recognition. Inhibitory effects of peptide analogs on the Grb2 SH2 domain and their binding affinities for Grb2 SH2 were determined by ELISA, cell-based assays, or Surface Plasman Resonance (SPR) technology. Results of theses studies provide important information for further modifications of lead peptides, and should lead to the discovery of potent peptides as anticancer agents.

Binding potency of peptide fragments of type 1 collagen cross-linked N-telopeptide measured by an enzyme-linked immunosorbant assay.

Osteoporosis represents a major healthcare problem affecting elderly person. Urinary level of the crosslinked N-telopeptide of type I collagen is a sensitive marker of bone resorption. Ten overlapping peptides covering the N-telopeptide of alpha-2 type I collagen were synthesized, purified, and assayed for their relative binding response to anti-type I collagen cross-linked N-telopeptide (NTX) antibody by using a competitive-inhibition enzyme-linked immunosorbent assay (ELISA). Peptides 1, 2, and 3, containing the N-terminal sequence of N-telopeptide, showed higher binding potency than peptides 4-10, suggesting that these peptides may contain binding sites for anti-NTX antibodies, and can serve as the lead for further preparation of their antibodies in order to develop novel bioassays for monitoring the bone loss in humans.